Methods are known for detecting specific nucleic acids or analytes in a sample with high specificity and sensitivity. Such methods generally require first amplifying nucleic acid sequence based on the presence of a specific target sequence or analyte. Following amplification, the amplified sequences are detected and quantified. Conventional detection systems for nucleic acids include detection of fluorescent labels, fluorescent enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels.
One disadvantage of these methods is that the labeled product not only requires some type of separation from the labeled starting materials but also, since the label is attached to the product, it is different than the natural product to be identified. It would, therefore, be of benefit to use methods and substrates that form unmodified product and at the same time generate a signal characteristic of the reaction taking place. It is of further benefit if the signal generated doesn't require separation from the starting materials but even if a separation is required the benefits of generating unmodified product in many cases are overwhelming.
Terminal-phosphate labeled nucleotides provide the above benefits. For example, incorporation of gamma- or delta-labeled nucleotides into DNA or RNA by nucleic acid polymerases results in the production of unmodified DNA or RNA and at the same time the labeled pyrophosphate generated can be used to detect, characterize and/or quantify the target. If these could be used in amplification reactions not only would they provide useful tools for detection and quantification of target sequence, but the amplified product, which is exact copies of the target sequence without modifications can be used in further studies.
DNA amplification by a number of amplification methods is performed at high temperatures. For example, in PCR, repeated cycles of denaturation at 95° C., annealing around 60° C. and extension around 70° C. causes significant breakdown of the dNTP's. This may significantly affect the yield of product in later cycles. Other amplification methods such as RCA and NASBA, although isothermal, also are conducted at higher temperatures. In case of NASBA, which is performed at 41° C., the stability of nucleotides may not be very critical, however in RCA which may be conducted at higher temperature depending upon the polymerase used and the complexity of sequence to be amplified, stability of nucleotides can be an issue under these conditions. If breakdown of the terminal-phosphate labeled nucleotides were to occur, the amount of background generated would overwhelm any signal directly related to the amplification process. It is therefore desirable to have nucleotides that can survive this repeated cycling of temperature or prolonged heating at a constant yet high temperature and hence continue to give high product yields and low background even in later cycles of amplification and possibly cut down the number of cycles/time required to achieve desirable amplification. Additionally, gamma-phosphate labeled nucleotides are extremely poor substrates for polymerase under the conditions normally used for nucleic acid synthesis and amplification. Synthesis of long stretches of nucleic acids (several hundred to several thousand bases long) would require hours if not days per cycle. Harding et. al. (WO 0244425 A2) describe the use of aminonaphthalenesulfonate-gamma-amido-dATP for DNA synthesis at high temperature. However, according to the inventors, in this case the synthesis only proceeds after the aminonaphthalenesulfonate hydrolyzes off the nucleotide and it is dATP that is used by the polymerase to form DNA. This of course is useless for detection or quantification of target sequence as the dye generated is independent of DNA synthesis.
A number of real time assays have been developed for quantification of DNA. Most of these can be classified into two categories. First category which is relatively easy to use involves the use of intercalating dyes, which have enhanced fluorescence upon intercalation. A number of nucleic acid stains such as ethidium bromide, SYBR Green® dyes, PicoGreen®, YOYO®, TOTO® or analogs have been developed as intercalators for real time assays. These, however, generate significant background signal partially due to intercalation between primer dimers and partially because they are fluorescent, albeit weakly, even when they are not intercalated.
The other category of real time assay is based on the use of fluorescence resonance energy transfer between a dye and a quencher. A number of these assays have been developed using FRET probes and or primers, such as Taqman, MGB Eclipse™, Scorpion primers, Molecular Beacons, sunrise primers, to name a few. These probes/primers are quenched by energy transfer until the amplification takes place and the quencher is physically separated from the dye or cleaved. Sensitivity of these assays depends greatly on the probe design and require a lot of optimization. In addition even with the best optimized probe, complete quenching is not achieved. So these assays can only provide a few fold enhancement in signal upon amplification and in the initial cycles background signal is much higher than the true signal.
It would be of benefit, therefore, to develop methods of amplification using terminal-phosphate labeled nucleoside polyphosphates where the amplification can be performed in reasonable time (similar to unmodified dNTP's) and the amount of label generated is proportional to the product formed. It is further desirable to have a real time assay, where the amount of label generated can be independently detected without interference of signal from the terminal-phosphate labeled nucleotide. It would be desirable to have a real time assay where the label is completely dark until the amplification proceeds.